EFFECTS OF HEME-L-ARGINATE ON L-NAME INDUCED HYPERTENSION. A Thesis Submitted to. The College of Graduate Studies & Research

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1 EFFECTS OF HEME-L-ARGINATE ON L-NAME INDUCED HYPERTENSION A Thesis Submitted to The College of Graduate Studies & Research In Partial Fulfillment of the Requirements For the Degree of Master of Science In the Department of Physiology University Nina Jean Lane Copyright Nina Jean Lane, August All Rights Reserved i

2 PERMISSION TO USE STATEMENT In presenting this thesis, I agree that the libraries of the University of Saskatchewan may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by Dr. Joseph Ndisang or, in his absence, by the Head of the Department of Physiology or the Dean of Medicine. It is understood that any copying, publication, or use of this thesis or any part for financial gain shall not be allowed without my expressed written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use that may be made of any material of this thesis. Requests for permission to copy or make other use of material in this thesis should be addressed to: Head of Department of Physiology College of Medicine University of Saskatchewan, 107 Wiggins Rd Saskatoon, Saskatchewan, Canada S7N 5E5 i

3 ABSTRACT N-ω-nitro-L-arginine methyl ester (L-NAME) has been used to induce experimental essential hypertension characterized by stimulation of the renin-angiotensin system (RAS) and oxidative system. Although the heme oxygenase (HO) system is known to suppress hypertension and the RAS, its effects on L-NAME-induced hypertension are poorly understood. Therefore, this study investigates the effects of heme-l-arginate (HA), a HO inducer, on L-NAME induced hypertension. HA (15mg/kg/day) was administered for 4 weeks either during the development or after the establishment (4 weeks) of L-NAME-induced (60mg/kg/day) hypertension in Sprague- Dawley (SD) rats. Vehicle control groups were used. Co-treatment with HA prevented the development of L-NAME induced hypertension, (124 mmhg, n=13 vs 168; n=10, 11 weeks; p<0.05). After L-NAME-induced hypertension was established for 4 weeks, HA therapy reduced blood pressure to normotensive at 15 weeks (123 mmhg, n=8 vs. 190 mmhg, n=7; (p<0.01). The prevention of hypertension was associated with increased HO-1 expression at 11 weeks (92.2±9.8 vs 15.9±9.9 HO-1/GAPDH %, n=4; p<0.01), reduction of heart Ang-II at 11 and 15 weeks (2.13±0.4 pg/mg, n=6 vs 4.06±0.4 pg/mg, n= 8; p<0.05 and 3.45±0.2 pg/mg, n=7 vs 4.23±0.2, n=7; p<0.05), respectively. HA co-treatment increased total antioxidant capacity (TAC) in heart tissue, the mesenteric artery and kidney. We conclude that up-regulating the HO system with HA normalizes blood pressure and prevents the development of L-NAME induced hypertension by suppressing Ang-II and abating oxidative stress. HA may be explored in the prevention and management of other forms hypertension characterized by elevated Ang-II and excessive oxidative stress. ii

4 ACKNOWLEDGEMENTS Firstly, I want to thank God for every opportunity granted to me and the abilities to complete my studies. I would like to thank my friends and family for their support and encouragement during the completion of my program, especially to my husband and my parents. I am grateful for the opportunities given to me Dr. Joseph Ndisang. I appreciate his direction and patience throughout my studies. I would like to thank the members of my advisory committee, Dr. Michel Desautels, Dr. Jane Alcorn, and Dr. Nigel West as well as Dr. Shah Amed, for their guidance and support throughout my graduate program. I am especially grateful for the support offered by the staff of the physiology department including Evelyn Bessel, Carol Ross, Gladys Weibe, Maureen Webster, and Dilip Singh. Support offered by many others throughout my research was invaluable: Dr. Ashok Jadhav, James Talbot and the research animal technicians in the animal quarters. Thank-you, to the Heart and Stroke Foundation for providing funding for this research. Lastly, I would like to extend my gratitude to Dr. Jim Thornhill and the College of Medicine for providing supplementary funding which allowed me to further my research. I dedicate this thesis to my two loves: husband, Jason Lane and my son, Connor Lane. The love, support and encouragement they gave allowed me to persevere and complete this program. iii

5 TABLE OF CONTENTS PERMISSION TO USE i ABSTRACT ii ACKNOWLEDGEMENTS iii TABLE OF CONTENTS iv LIST OF FIGURES vii LIST OF TABLES viii LIST OF ABBREVIATIONS ix 1. INTRODUCTION Hypertension Overview Essential Hypertension The role of the sympathetic nervous system in essential hypertension The role of endothelial dysfunction in essential hypertension The role of renin-angiotensin system in essential hypertension Angiotensin Oxidative Stress Remodeling, Hypertrophy, Inflammation Animals Models of Hypertension Spontaneously Hypertertensive Rat TGR(Ren2)27 Hypertensive Rat The Goldblatt Hypertensive Model Deoxycorticosterone acetate-salt Hypertensive Model N-ω-nitro-L-arginine methyl ester (L-NAME)-induced Hypertension Mechanisms of L-NAME induced Hypertension Current Therapies used to counteract L-NAME induced Hypertension The Heme Oxygenase System Review of the Heme Oxygenase System Carbon Monoxide Biliverdin/Bilirubin Free Iron Role of the Heme Oxygenase in Hypertension Implications of heme oxygenase inducers as treatment in L-NAME induced hypertension RATIONALE, HYPOTHESIS AND OBJECTIVE Rationale Hypothesis Objective 28 iv

6 2.3.1 To determine the effect of heme-l-arginate on the development of L-NAME induced hypertension To determine the effect of heme-l-arginate on established of L-NAME induced hypertension MATERIALS AND METHODS Animal Care and Handling Experimental Design Treatment Protocol Preparation of Solutions Preparation of L-NAME Preparation of Heme-L-Arginate Measurment of Systolic Blood Pressure Recording of Food intake, Fluid Intake and Urine output Assessment of Body and Organ Weight Determination of HO-1 expression via Western Blot analysis Quantification of Angiotensin II levels Determination of total antioxidant capacity Statistical Analysis RESULTS Effect of heme-l-arginate on systolic blood pressure in L-NAME induced hyperstension The effect of heme-l-arginate of food intake, water intake and urine excretion on L-NAME induced hypertension Assessment of gross body and organ weight The effect of heme-l-arginate on the expression of HO-1 in the heart Modulation of Angiotensin II by L-NAME and heme-l-arginate Effects of heme-l-arginate on the total antioxidant capacity DISCUSSION The consequences L-NAME-induced hypertension and the benefits of induction of the heme oxygenase pathway Induction of hypertension after L-NAME administration Modulation of systolic blood pressure in L-NAME induced hypertension with heme-l-arginate Heme-L-arginate upregulates expression of HO-1 57 v

7 5.5 Alterations of Angiotensin II levels by L-NAME and heme-l-arginate Antioxidant status altered by heme-l-arginate in L-NAME induced hypertension Heme-L-arginate altered body weight and fluid balance, but not food intake or wet heart weight Limitation of Study CONCLUSIONS PERSPECTIVES FUTURE DIRECTIONS REFERENCES 68 vi

8 LIST OF FIGURES Figure 1.1. N-ω-nitro-L-arginine methyl ester modulation of blood pressure 19 Figure 3.1. Study time line pressure 31 Figure The effect of heme-l-arginate on systolic blood pressure of L-NAME -induced hypertension 38 Figure The effect of heme-l-arginate on systolic blood pressure in established L-NAME-induced hypertension 39 Figure Food and water intake in rats treated with L-NAME and Heme-L -arginate 41 Figure The effect of L-NAME and Heme-L-arginate on urine output 42 Figure The effect of Heme-L-arginate on food intake in L-NAME induced hypertension 46 Figure The effect of heme-l-arginate and L-NAME on heme-oxygenase 1 (HO-1) protein expression against GAPDH using representative western blot and densitometry analysis in heart tissue 47 Figure The effect of heme-l-arginate and L-NAME on Angiotensin II in heart and plasma 49 Figure The effect of heme-l-arginate and L-NAME on Total Antioxidant Capacity in the heart 51 Figure The effect of heme-l-arginate and L-NAME on Total Antioxidant Capacity in kidney and mesenteric artery 52 vii

9 LIST OF TABLES Table 1 The effect of heme-l-arginate and L-NAME on body and organ weight 44 viii

10 LIST OF ABBREVIATIONS AA ABTS AC arachidonic acid 2,2 -azino-di-[3-ethylbenzthiazoline sulphonate adenylate cyclase ACE1 angiotensin converting enzyme type 1 ACE2 angiotensin converting enzyme 2 Ang (1-7) angiotensin (1-7) Ang I Ang II ANOVA AT 1 ATP BK Ca BVR Ca 2+ camp cgmp CO COX cp450 CRP DAG DiHEET angiotensin I angiotensin II analysis of variance angiotensin type 1 receptor adenosine triphosphate calcium activated potassium channel biliverdin reductase cytosolic free calcium cyclic adenosine monophosphate cyclic guanosine monophosphate carbon monoxide cyclooxygenase cytochrome P450 C reactive protein diacylglycerol dihydroxyeicosatetraenoic acids ix

11 DOCA ECM EDCF EDRF EET EIA ET enos g GAPDH GTP HA HCl HEET HO H ICAM inos ip IP 3 IRP K + NaOH N-LAME deoxycorticosterone acetate extracellular matrix endothelial derived contracting factors endothelial derived relaxing factor epoxyeicosatetraenoic acids enzyme immunoassay endothelin endothelial nitric oxide synthase grams glyceraldehyde-3-phosphate dehydrogenase guanosine triphosphate heme-l-arginate hydrochloric acid hydroxyeicosatetraenoic acids heme-oxygenase hydrogen peroxide intracellular adhesion molecules inducible nitric oxide synthase intraperitoneal inositol triphosphate iron regulatory protein potassium sodium hydroxide N-ω-nitro-L-arginine methyl ester x

12 M mab MAPK mg/kg/day mmhg NAD(P)H NO NFκB O 2ˉ ONNO - PBS PDE PKA PKG molar monoclonal antibody mitogen-activated protein kinase milligrams/kilogram/day millimeters of mercury nicotinamide adenine dinucleotide phosphate nitric oxide nuclear factor κb superoxide anion peroxynitrite phosphate buffered saline phosphodiesterase protein kinase A protein kinase G PLA 2 phospholipase A 2 PLC RAS ROS rpm SBP SD SEM sgc SHR phospholipase C renin-angiotensin system reactive oxygen species revolutions per minute systolic blood pressure Sprague Dawley standard error of the mean soluble guanylyl cyclase spontaneously hypertensive rat xi

13 SNS SOD SP-SHR TAC TEAC sympathetic nervous system superoxide dismutase stroke prone-spontaneously hypertensive rats total antioxidant capacity Trolox equivalent antioxidant capacity TGR(Ren2)27 transgenic rat (Ren2)27 TNFα VCAM VSMC 1K1C 2K1C 2K2C tumor necrosis factor α vascular cell adhesion molecule vascular smooth muscle cell one-kidney one-clip two-kidney one-clip two-kidney two-clip xii

14 1. INTRODUCTION 1.1 Hypertension Overview Blood pressure is a result of many interconnected factors. It is the product of cardiac output and total peripheral resistance, while cardiac output is the product of stroke volume and heart rate [1]. Nervous and endocrine systems as well as genetic and environmental factors work together in a complex system to determine an individual s blood pressure [2-4]. A measure of systolic and diastolic normal blood pressure is considered to be equal or less than 120/80 millimeters of Mercury (mmhg); in humans hypertension is defined as blood pressure equal to or above 140/90 mmhg [5]. Hypertension is considered to be a major risk factor for an array of cardiovascular and associated diseases, including heart failure, peripheral arterial disease and end stage renal disease [6]. Hypertensive states have also been linked to increased risk for type 2- diabetes and cardiometabolic syndrome [7, 8]. In fact, numerous studies have found a continuous relationship between elevated systolic and diastolic blood pressure and cardiovascular morbidity and mortality [5]. Early interventions and control of elevated blood pressure result in improved health outcomes [9]. However, despite current research and the large number of available treatments, hypertension remains a problem in both developed and developing nations and it is now being diagnosed in adolescents and children [10]. Corresponding with the aging population, Kearney reports that 26.4 % (972 million) of the world s population over the age of 20 had hypertension in 2000 and that will increase to 29.2% (1.56 billion) by 2025 [11]. Hypertension is a growing global problem and despite the decades of research its incidence continues to grow calling for more elucidation of its mechanisms and potential treatments. 1

15 1.2 Essential Hypertension The most common and puzzling form of hypertension is essential or primary hypertension. Historically named essential hypertension due to the theory that increases in total peripheral resistance and subsequently blood pressure were essential to aging. Presently, essential hypertension is defined as the rise of blood pressure due to an unknown cause [12]. Even more startling, essential hypertension is thought to account for up to 95% of hypertension cases [13]. While the complexities of its pathogenesis are not fully understood, it is considered to be multi-faceted involving neurogenic stimuli, endothelial dysfunction and the renin-angiotensin system (RAS) resulting in oxidative stress, remodeling and inflammatory processes [14, 15]. Moment to moment regulation of hypertension is regulated by neurogenic systemic peripheral vasoconstriction driven by the sympathetic nervous system (SNS), quickly resulting in endothelial dysfunction and activation of the RAS [16] The Role of the Sympathetic Nervous System in Essential Hypertension The sympathetic nervous system (SNS) is one component of the autonomic nervous system. The other component, the parasympathetic nervous system innervates the heart and a small number of blood vessels, while the SNS provides widespread direct and indirect control of cardiac and vascular function, innervating the heart, blood vessels, adrenal gland and kidneys. Responsible for the transient flight or fight response, post-ganglionic sympathetic neurons release norepinephrine resulting in activation of the adrenergic pathways [17]. Directly connected to this response is the adrenal medulla, with its chromaffin cells acting as postsynaptic ganglion sympathetic neurons releasing epinephrine. The α 1 -adrenoreceptors stimulate vasoconstriction and elevation of heart rate as well as stimulating the RAS by causing the release of renin [17]. Presynaptic α 2 -adrenoreceptors provide a negative feedback response to the α 1-2

16 adrenoreceptors by inhibiting the release of norepinephrine, while post-synaptic α 2 - adrenoreceptors stimulate platelet aggregation and vasoconstriction [18]. In addition to the α- adrenoreceptors, the β-adrenoreceptors also contribute to the regulation of blood pressure. β 1 - adrenoreceptor activation increases heart rate and contractility, while β 2- adrenoreceptors activation promotes vasodilation [19]. Moreover, under normal conditions the SNS rapidly responds to increases and decreases in blood pressure via arterial baroreceptors augmenting total peripheral resistance, heart rate and cardiac contractility [20]. The SNS also augments arterial blood pressure in response to chemoreceptors acting in response to the blood oxygen-carbon dioxide balance [20]. In fact, moment to moment changes in blood pressure are almost entirely mediated by the nervous system [21]. While the majority of anti-hypertensive research has focused on RAS targeting therapies, general consensus is that SNS over-activity is involved in the initiation and may contribute to sustaining essential hypertension [2]. SNS over-activity has been proven to cause elevated arterial pressure, the precise mechanism leading to the prolonged SNS over-activity is unclear [22]. Before the availability of antihypertensives clinicians recognized the importance of SNS in the control of long term blood pressure, using sympathectomies to reduce hypertension [23]. SNS over-activity has been directly involved in the increased morbidity in a number of pathophysiological conditions such as heart failure and end stage renal disease [24]. Working in conjunction with SNS activity, stimulation of endothelial activity and subsequent dysfunction contribute to the development and maintenance of essential hypertension The Role of Endothelial Dysfunction in Essential Hypertension Endothelial dysfunction is defined as a functional and reversible alteration of endothelial cell relaxation, and while resulting from interplay of many factors, is often due to impaired nitric 3

17 oxide (NO) availability [25, 26]. Known as the primary endothelial derived relaxing factor (EDRF), the majority of the endothelial-dependent vasodilation has been attributed to NO [27]. Synthesis of NO from the oxidation of L-arginine by endothelial or inducible derived nitric oxide synthase (enos or inos, respectively) is stimulated by mechanical factors, such as pulsatile and shear stress, and hormonal signals [28]. In the blood vessels, NO binds to the intracellular enzyme soluble guanylyl cyclase (sgc), which then leads to a 200-fold increase in cyclic guanosine monophosphate (cgmp) from guanosine triphospate (GTP) [29]. cgmp then acts on protein effectors including; protein kinase G (PKG), cyclic adenosine monophosphate (camp)- dependent protein kinase A (PKA), cyclic nucleotide-gated cation channels and phosphodiesterase (PDE) [28, 30]. The binding of cgmp to these proteins affects several physiological processes for instance, vascular relaxation and dilation, cardiac systole and immune mediated inflammation [30]. NO also acts independently of cgmp contributing further to vasodilation as well as regulating apoptosis, remodeling and angiogenesis; consequently, NO produced in the vasculature plays an important role in maintaining blood pressure [31]. In fact, inhibition of NOS results in elevation of peripheral vascular resistance, activation of the SNS and increased arterial blood pressure associated with subsequent structural and functional changes [32, 33]. Reduced NO itself has not only been linked to endothelial dysfunction, but to arterial stiffness, and in contrast a number of therapies have been shown to improve NO, endothelial function and reducing arterial stiffness [34]. In contrast to NO, endothelial derived contracting factors (EDCF) oppose the vasodilation of NO and exacerbate endothelial dysfunction. Endothelin (ET) is one of the most potent EDCF and is released continuously from endothelial cells [35]. Found in three isoforms, ET activates two subtypes of G-protein receptors leading to the formation of inositol triphosphate (IP 3 ) and subsequent vasoconstriction [36]. ET-1 in particular not only regulates vasoconstriction, but is 4

18 important in regulating processes such as remodeling, angiogenesis and extracellular matrix (ECM) synthesis [37]. ET-1 has been connected to both pulmonary and systemic hypertension. In fact, peripheral endothelial dysfunction has been thought to contribute to SNS over-activity and result in arterial hypertension [38]. Strong evidence supports the contribution of endothelial dysfunction to the development of essential hypertension. Endothelial dysfunction and blood pressure are further modulated by the activity of the RAS and its mediated effects as discussed below The Role of the Renin-Angiotensin System in Hypertension Hormones play a crucial role in the regulation of blood pressure, including; vasopressin, aldosterone and the RAS. The RAS is a crucial effector of not only the cardiovascular system but of several organ systems. RAS controls blood pressure both through vascular tone and fluidelectrolyte balance. The synthesis and secretion of the hormone renin is the rate limiting step in the mobilization of the RAS [39]. Renin-synthesizing cells are found in the adult kidney where they respond to intrarenal baroreceptors, the sodium chloride load at the macula densa, SNS activity via β-adrenoreceptor activation and angiotensin II (Ang II) with interplay of second messengers, including: camp, cgmp and free cytosolic calcium [40, 41]. Activation of camp leads to phosphorylation of PKA or inhibition of PDE. Inhibition of PDE is responsible for the degradation of camp, and is a stimulator of renin secretion [39, 40]. In fact, many established activators of AC can result in increased renin secretion including activators of adrenoreceptors (catecholamines), and hormones, for example prostaglandins E 2 and I 2, and dopamine [40]. Renin levels are further modulated due to cgmp stimulated NO and subsequent inhibition of PDE via slowing of renin degradation [42]. Interestingly, cgmp can also inhibit renin secretion 5

19 rates through activation of a protein kinase [43]. Increases in cytosolic free calcium (Ca 2+ ) strongly inhibit renin release [44]. Decreases in glomerular filtration rate stimulate renin secretion, indicating pressure regulation at the renal level [39]. Over stimulation of renin synthesis and secretion is a primary cause for increased blood volume, hypertension and organ damage. Not only is renin the rate limiting enzyme of the RAS, it activates the pathway for the formation of the primary effecter peptide of the RAS, Ang II. Renin cleaves angiotensinogen, which primarily originates in the liver, to form angiotensin I (Ang I). Angiotensin converting enzyme type 1 (ACE1), the primary form of ACE, is produced predominantly from lung endothelium and hydrolyzes Ang I to form Ang II, an octapeptide, contributing to a vast array of actions. Interestingly, ACE1 also has the ability to hydrolyze bradykinin and kallidin, both known to lower blood pressure [45]. However, the net effect of ACE1 is to increase blood pressure by increasing a vasoconstriction [46]. In contrast to ACE1, angiotensin converting enzyme type 2 (ACE2) is an enzyme that primarily acts on Ang II. Found in several tissues, ACE2 cleaves Ang II resulting in the production of the heptapeptide angiotensin (1-7) (Ang (1-7) [39]. Ang (1-7) is an antagonist to Ang II and a potentiator of vasodilation through stimulation of prostaglandins, NO and bradykinin [47]. It should be noted that another enzyme, chymase, also acts on Ang I to form Ang II, but to a much lesser extent [48] Angiotensin II and Hypertension Ang II is the primary effector peptide of the RAS and controls much of the pressor, angiogenic and remodeling effects of the RAS [49]. Studies show that Ang II is distributed systemically as well as locally in tissues such as heart and kidney [50]. There are four known angiotensin receptors, but most actions of Ang II are mediated through G protein-coupled angiotensin type 1 (AT 1 ) receptor [46]. Through AT 1, Ang II exerts a multitude of effects which 6

20 contribute to hypertension including intermediate acting vasoconstriction, sodium reabsorption and stimulation of aldosterone synthesis [51]. Short term, Ang II is responsible for regulation of vascular tone through vasoconstriction [52]. The presence of Ang II immediately induces vasoconstriction via G proteins resulting in the activation of phospholipase C (PLC), leading to the hydrolysis of phosphatidyl inositol and subsequent formation of IP 3 and diacylglycerol (DAG) [53]. IP 3 results in increases in Ca 2+ concentration by causing increased Ca 2+ influx mobilizing intracellular Ca 2+ the primary trigger for vascular contraction [54]. DAG together with Ca 2+ activates PKC, further promoting vasoconstriction and vascular smooth muscle cell (VSMC) growth [55]. PKC mediated Ang II signaling also stimulates vasoconstriction via a sodium/hydrogen exchanger which changes intracellular ph (alkalinization) and modulates an actin-mysoin interaction [54]. PKC carries out its actions through phosphorylation of tyrosine kinases and mitogen-activated protein kinase (MAPK) signaling pathways [52]. Ang II also activates phospholipase A 2 (PLA 2 ) and the subsequent release of arachidonic acid (AA). AA is processed into many different eicosanoids by cyclooxygenase (COX), lipoxygenase or cytochrome P450 (cp450), influencing many vascular and renal actions [56]. For instance, COX metabolism of AA yields unstable and short lived endoperoxides which cause downstream generation of thromboxane A 2 and prostacyclin, leading to thrombosis or anti-platelet aggregation, respectively [57] Moreover, cp450 metabolizes AA to epoxyeicosatrienoic acids (EETs), dihydroxyeicosatetraenoic acids (DiHETEs) and hydroxyeicosatetraenoic acid (HETE), most notably 19 and 20 [58]. While EETs are vasodilators, 20-HETE is produced in VSMC and is a potent vasoconstrictor and promoter of angiogenesis [59]. HETEs are vasoconstrictors which decrease renal sodium excretion and increase fluid retention increasing blood pressure further [60]. 7

21 Ang II further regulates the fluid-electrolyte balance. Within the kidney, Ang II increases reabsorption of sodium and fluid in the proximal and distal tubules directly along with augmentation of the glomerular filtration rate [61]. Ang II also stimulates thirst and appetite for salt by acting on the hypothalamus [41]. Under normal physiological conditions Ang II increases glomerular permeability in the kidneys; however, it can also cause inflammation leading to tubulointerstitial damage and proteinuria of the kidneys [62]. Indirectly, Ang II also contributes to sodium and fluid reabsorption through stimulation of synthesis of a steroid hormone with mineralocorticoid activity, aldosterone [63]. Stimulation of mineralocorticoid receptor by aldosterone gives rise to the expression of proteins that stimulate multiple sodium transport mechanisms including a sodium-potassium adenosine triphosphate (ATP) pump leading to increased sodium and fluid retention in the distal tubule [48]. Mainly formed in the adrenal glomerulosa, aldosterone can exert effects on the cardiovascular and renal systems because of the wide distribution of its cytoplasmic mineralocorticoid receptor [63]. Recent studies show that aldosterone also promotes remodeling and inflammation of the vasculature [64]. Outside of its fluid balance role, aldosterone independently contributes to the long term deleterious effects of Ang II with the production of reactive oxygen species, inflammation, remodeling and fibrosis [65-67] Oxidative Stress and Hypertension While many of Ang II s damaging effects are direct, growing evidence indicates that a key component of Ang II mediated effects is generation of reactive oxygen species (ROS) [51, 68, 69]. Sustained over production of ROS and subsequent imbalance between oxidants and antioxidant capacity are considered to be oxidative stress [70]. All vascular cell types have the ability to produce ROS, which are associated with inflammatory responses [71]. While any 8

22 electron-transferring protein or enzyme can produce ROS, it is emerging that vascular ROS is primarily produced through the stimulation of vascular nicotinamide adenine dinucleotide phosphate (NAD(P)H)-oxidase with contributions from xanthine oxidase and uncoupled enos [15, 72]. NAD(P)H-oxidase is found in all vascular cell types, for instance endothelial cells, fibroblasts and VSMC [69]. Non-phagocytic NAD(P)H)-oxidase is regulated by many factors, including; hormones, growth factors and mechanical stimuli, but the pathway that is best characterized is regulation via AT 1 receptor activation by Ang II [72, 73]. Significantly, well unclear of exact mechanisms studies show that AT 1 receptor is activated in patients with essential hypertension and plays a role in ROS over-production [74]. Also, recent studies have indicated that hypertension with elevated endogenous levels of Ang II, resulting from RAS activity, resulted in increased NAD(P)H-derived ROS [54, 73]. Moreover, studies of animal models of Ang II-induced hypertension have revealed elevated expression of several NAD(P)H-oxidase subunits and increase activity of NAD(P)H-oxidase [62, 73]. Vascular NAD(P)H-oxidase activation, the major source of ROS in hypertension, results in the reduction of molecular oxygen to form superoxide anion (O 2ˉ) and a contributing source of hydrogen peroxide (H ) [15, 68, 71]. O 2ˉ produced from the NAD(P)H-oxidase is unstable and is quickly reduced or oxidized [68]. O 2ˉ is considered one of the most potent ROS since it causes significant vascular injury as well as promotes damage carried out by an array of secondary products. The effects of O 2ˉ are thought to be local through internal signalling because it is hydrophilic and has a negative charge. One study showed Ang II-induced hypertension led to the doubling of O 2ˉ formed by NAD(P)Hoxidase [73]. In vascular endothelium, xanthine oxidase also contributes to the enzymatic formation of O 2ˉ [15]. In the endothelium, subsequent oxidization of O 2ˉ yields peroxynitrite (ONNO - ) by scavenging NO [71]. ONNO - is a strong oxidant, which has the ability to cause cell 9

23 damage by oxidizing proteins, lipids and nucleic acids [75]. Specifically, ONNO - has been shown to uncouple enos and inos, augmenting the antioxidant producing enos into a ROS producing enzyme, furthering the oxidative stress and activating the SNS [33, 76]. Interestingly, aldosterone further promotes NAD(P)H-oxidase activity through decreased protein expression as well as endothelial NOS uncoupling exacerbating the effects of Ang II [64]. Reduction of O 2ˉ by superoxide dismutase (SOD) results in the production of H H is a more stable ROS and has been implicated in the reduction of endothelial NOS and along with ONNO - contributes to increased oxidative stress [68, 77]. It is also an important ROS because of its lipophilic nature allowing it to cross cell membranes leading to a widespread effect. Also contributing to oxidative stress, the hydroxyl radical is a potent oxidant with an extremely short half life. It can be produced directly from water [78]. The production of these Ang II-mediated ROS influences inflammatory signaling pathways and related molecules leading to overall reduction in endogenous antioxidants [15]. In fact, hydroxyl radical scavengers are known to augment responses to Ang II in SHR [79]. Evidence suggest that ROS contribute significantly to the development of pathophysiological conditions such as hypertension, cardiovascular disease and renal damage [70]. It is important to note that the amount of oxidative damage is directly related to the availability of antioxidants. Antioxidants can be enzymatic or non-enzymatic in nature. One such enzymatic antioxidant is SOD. SODs are metalloenzymes whose role in the antioxidant defense mechanism is catalyzing the dismutation of the superoxide anion to molecular oxygen and H at the cellular level. There are three isoforms of SODs in mammals: cytosolic and extracellular copper-zinc SOD and mitochondrial manganese SOD [80]. Copper-zinc SOD is the dominant isoform with high levels of expression in all cells and in particular vascular tissue [81]. Mitochondria are known producers of O - 2 and as such manganese SOD is a first line of defense 10

24 against oxidation. Catalase works in conjunction with SOD converting H to benign oxygen and water [82]. Similar to catalase, glutathione peroxidase scavenges H by oxidation of glutathione to glutathione disulfide and reduces it to water. Importantly, glutathione peroxidase is also able to reduce lipid peroxides and lipid alcohols. Non-enzymatic antioxidants are crucial in the combating of oxidative stress. They include ascorbic acid, α-tocopherol, carotenoids, α-lipoic acid, polyphenols and tetrohydrocurcumin. In conjunction these antioxidants significantly protect against ROS and their damaging effects and have been noted to reduce hypertension, oxidative induced remodeling and atherosclerosis. The balance of ROS between antioxidants is very important for prevention of disease. With elevation of ROS in the cardiovascular system the resultant receptor activation, modulation of transcription factors and protein expression, leads to deleterious effects including vascular remodeling, endothelial dysfunction and hypertrophy [62, 72, 83] Remodeling, Hypertrophy, Inflammation and Hypertension Chronic hypertension can result remodeling, hypertrophy and increased total peripheral resistance [51, 84]. Vessels are composed of cells and ECM that are dynamic. Remodeling is associated with the reorganization of VSMC, changes in ECM composition and elastic fiber alteration [12]. While remodeling is considered a normal adaptive response to increased wall stress, it also plays a role in the maintenance of hypertension. Ang II is a powerful mediator of vascular remodeling through the stimulation of inflammatory molecules, growth factors and chemokines [85]. Ang II stimulates molecules such as prostaglandins and fibronectin, which are expressed in a variety of cardiovascular tissue including: VSMC, endothelial cells, cardiac fibroblasts and mesangial cells [86, 87]. Fibronectin, an ECM protein, binds to collagen and modulates fibrillogenesis, increasing collagen type 1 and decreasing collagen type 2 which are 11

25 responsible for stiffness and elasticity, respectively [88]. Furthering inflammation, Ang II enhances cellular adhesion through chemokines and cytokines. Essential to Ang II-mediated effects, nuclear factor κb (NFκB) is particularly important in vascular inflammation, VSMC proliferation and migration as it controls many proinflammatory genes. Ang II, and Ang II-resultant ROS such as O 2ˉ and H 2 0 2, stimulate nuclear translocation, DNA binding and transcription of a NF-κB gene as well as protein expression of growth factors (tumor necrosis factor α (TNFα)), adhesion molecules [85, 89]. For example, selectins make the initial contact, leukocyte recruitment into the vessel wall is regulated through the stimulation of adhesion molecules, intracellular adhesion molecules (ICAM) 1 and 2 and vascular cell adhesion molecule-1 (VCAM-1) [62, 90]. The increase of ICAM-1 and VCAM-1 also involve activation of MAPK pathways, which are regulated by oxidation [91]. Also partially upregulated by H 2 0 2, MAPK growth signaling pathway is well characterized and leads to phosphorylation of proteins and subsequently influence cell cycle, apoptosis, differentiation and ultimately hypertrophy [90, 92]. After leukocyte recruitment takes place, chemokines play a key role in the migration of leukocytes into tissues [90, 91]. Once leukocytes move in to tissues they release metabolites and proteases that are toxic and may promote tissue damage. In chronic hypertension, inflammation is characterized by progressive replacement of leukocytes to mononuclear cells, subsequently undergoing transformation to macrophages which are phagocytic mediators of the tissue destruction, vascular proliferation, and fibrosis [93]. There are many markers of inflammation and vascular remodeling but one of the most powerful and stable is thought to be C reactive protein (CRP) [62]. Higher prevalence of hypertension has been correlated with CRP levels and evidence has suggested that CRP may be an independent risk factor for hypertension [94]. CRP is a direct participant in the Ang IImediated response through the cascade production and stimulation of chemokines and adhesion 12

26 molecules [52]. Expression of CRP has been noted in VSMC and macrophages within atherosclerotic plaques [95]. In addition, CRP activation contributes to hypertension by inhibiting enos expression and activity [85]. The expression of CRP potentiates the actions of Ang II via increasing the expression of AT 1 [49]. The sum effect of the vasoconstriction, modulation of growth factors, adhesion molecule, and inflammatory process in hypertension is remodeling and hypertrophy. Remodeling is characterized by rearrangement of the ECM and VSMC, can be measured by a change in the ratio of medial thickness to lumen diameter. In hypertension, increased shear forces combined with the Ang II-mediated effects lead to vascular remodeling and ultimately atherosclerosis [96]. Hypertrophy refers to an increase size of cells or increase thickness of a vessel, without increases in cell number. Additional evidence linking Ang II to cardiovascular hypertrophy comes from studies of AT 1 receptor antagonists in animal models. Several AT 1 receptor antagonists have been reported to reduce left ventricular mass. By altering the functionality of tissues, remodeling and hypertrophy, stimulated by Ang II and its multitude of effects, leads to the development of sustained hypertension and a range of cardiovascular disorders and renal damage [51]. The severity and commonness of essential hypertension clearly indicate a need for further study on its driving mechanisms and possible therapies. 1.3 Animal Models of Hypertension Animal models have proved to be useful in elucidating cause and progression of hypertension. Hypertension is diverse as the methods used to induce it in animals. Variables such as food and fluid intakes, structural alterations, environment, pharmacological intervention and importantly genetics have been manipulated to induce experimental hypertension in animals. Many models help to elucidate the many causes of hypertension in humans, such as the 13

27 spontaneously hypertensive rat (SHR), transgenic rat (Ren2)27 (TGR(Ren2)27, Goldblatt and deoxycorticosterone acetate (DOCA) hypertension models Spontaneously Hypertensive Rat A genetic hypertensive model, the SHR is the most often used model of hypertension and it is the standard in research of essential hypertension [97]. Inbred from Wistar and Wistar- Kyoto non-hypertensive controls, SHR develop hypertension at 4-6 weeks of age without intervention [98]. In early stages of hypertension, SHR maintain total peripheral resistance, but have increased cardiac output. However, as hypertension is established hypertrophic vessels increase total peripheral resistance, cardiac output returns to normal and remodeling in the heart occurs [99, 100]. Like in human essential hypertension, the exact cause is unknown but SHR show changes in SNS activity, alterations in NO availability and endothelial dysfunction [101, 102]. Levels of sgc and cgmp were found to be significantly lower in young SHR compared with age-matched Wistar-Kyoto [103]. Increases in arterial wall renin have been observed in SHR. While increases in Ang II has not been noted, sensitivity to Ang II is seen in SHR [104] TGR(Ren2)27 Hypertensive Rat Like SHR, the (Ren2)27 TGR(Ren2)27 is an experimental model of essential hypertension that also shows sensitivity to Ang II. Created by Mullins et al., the TGR(Ren2)27 rat was developed through the introduction of the mouse Ren2 gene into rats [105]. It had been previously reported that injection of purified mouse renin elevated blood pressure and the Ren2 gene had shown high expression in a transgenic mouse model [106]. Homozygous TGR(Ren2)27 rats develop severe hypertension at an early age and reach maximum levels at 9 weeks of age. Similar to the SHR, the mechanisms behind the rise in blood pressure in the TGR(Ren2)27 rat 14

28 have not been clearly elucidated. Interestingly, research has noted that circulating renin levels remained unchanged or decreased as compared to heterozygous normotensive littermates [107]. Also, the adrenal gland of the TGR(Ren2)27 is hypertrophic along with large increases in local renin and aldosterone [108]. Ang II levels are found to be elevated, but more importantly the VSMC were shown to have increased sensitivity to Ang II, similar to observed changes in SHR [106]. In young, but not aged, TGR(Ren2)27 rats plasma steroid levels and secretion are enhanced and may be involved in the development of hypertension [108]. Aged TGR(Ren2)27 rats show diminished NO release suggesting endothelial dysfunction. The sum of these changes is remodeling, hypertrophy and end-organ damage. The development of TGR(Ren2)27 rats allow for further study of hypertension and its pathophysiology, but may not be representative of human hypertension due to its early on-set and severe nature [109]. As noted above, genetics play an important role in the development and sustaining of hypertension. However, structural changes and food and fluid intake are also integral factors in some forms of hypertension, as observed in the Goldbatt and DOCA animal models The Goldblatt Hypertensive Rat The first animal model of hypertension was developed by Harry Goldblatt in 1934 when he clipped the renal artery of a dog and produced a hypertensive state [110]. As an experimental model of renal and secondary hypertension, it can include one of the following: two-kidney oneclip (2K1C) where both kidneys remain intact and one renal artery in constricted with a clamp, one-kidney one-clip (1K1C) where one kidney is removed and the renal artery of the remaining kidney is clamped or two-kidney two-clip (2K2C) where both kidneys are intact but either the aorta or both renal arteries are clamped. The clamps reduce renal perfusion pressure which in turn stimulates renin and Ang II synthesis [109]. This results in endothelial dysfunction, 15

29 increased peripheral resistance and subsequent increases in blood pressure. Unlike the 2K1C, the 1K1C model is considered to be sodium-fluid volume dependent because of the absence of the normal kidney and its absent compensatory elevated sodium and water excretion, leading to fluid retention [111]. Additionally, the 2K2C exhibit severe renal ischemia, as well as both increased RAS and SNS activity [109]. In rats, the Goldblatt hypertensive model induces a chronic hypertensive state with increased renin and subsequent Ang II, similar to that in humans with unilateral renal artery stenosis [97]. In contrast to the high renin levels noted in the Goldblatt hypertensive models, the administration of DOCA induces a low renin form of hypertension [112] Deoxycorticosterone acetate-salt Hypertensive Model The DOCA-salt model uses synthetic mineralocorticoid steroids and sodium chloride to mimic aldosterone overload and induce volume overload and subsequent hypertension via retention of sodium and water [113, 114]. It is characterized by endothelial dysfunction, elevated RAS activity and oxidative stress [97, ]. DOCA-salt rats exhibit low renin and do not respond well to RAS inhibitors, such ACE inhibitors or Ang II antagonists [97]. However, DOCA-salt hypertension does respond well to diuretics and aldosterone inhibitors [118]. DOCAsalt rats develop severe hypertrophy and end-organ damage [109]. This low renin, volume overload model of hypertension is valuable to study because it mimics the outcome of chronic human essential hypertension. The DOCA-salt hypertensive model and many other animal models allow further elucidation of the many unknown mechanisms driving human hypertension. Notably, a key element in all the experimental models above is the alteration in Ang II or the modulation of sensitivity to Ang II. Similar to these models is N-ω-nitro-L-arginine methyl ester (L-NAME)-induced hypertension. 16

30 1.4 N-ω-nitro-L-arginine methyl ester (L-NAME)-induced hypertension Mechanisms of L-NAME-induced Hypertension Experimental models of hypertension are extremely important in the exploration of hypertension. One such established model is L-NAME hypertension, a pharmacologically induced form of experimental hypertension [119]. Routes of effective administration include intravenous, intraperitoneal and oral. L-NAME produces a hypertensive state reflective of the dysfunction seen in essential hypertension via several mechanisms, including: inhibition of NO, SNS activity increasing total peripheral resistance, oxidative stress and arterial remodeling (Figure 1.1) [38]. Classically, L -NAME is known as an inhibitor of NOS leading to decreased NO, an important vasodilator [26]. Acute and chronic inhibition of NO produces endothelial dysfunction, which has been clearly demonstrated by L-NAME [26]. Moreover, the inhibition of NO by L-NAME in young SHR produce similar results to that of naturally aged SHR, supporting the role for NO inhibition in the study of essential hypertension [120]. In addition to the reduction of NOS activity by L-NAME, the production of O 2ˉ by L-NAME may decrease NO further through its ONOO - -mediated uncoupling of NOS [67]. While controversial, it has been reported that L-NAME may directly alter baroreceptor sensitivity in SNS leading to augmentation of blood pressure [121]. It is hypothesized that NO is an inhibitory modulator of SNS outflow [122]. Large volumes of evidence show that stimulation of SNS activity in L- NAME-induced hypertension can be modulated by reduced NO availability [38]. Despite the method of induction, SNS activity is a contributor to acute and chronic L-NAME-induced hypertension [121, 123]. In addition to the attenuation of NO, evidence has also shown that L- NAME stimulates the RAS [26, 119]. 17

31 As in essential hypertension, RAS is stimulated at many levels by L-NAME. Studies of chronic L-NAME administration indicate increased mrna expression of renin and, consequently; elevated levels and activity of renin have been reported in the plasma [124, 125]. In addition, modulation of ACE, the enzyme responsible for the conversion of Ang I to Ang II, by L-NAME has been observed [126]. Further evidence reveals that as duration of L-NAME treatment increases, ACE activity in plasma and tissue increases accordingly [126, 127]. Both, antagonists of AT 1 receptor and inhibitors of ACE prevented the development of L-NAMEinduced hypertension revealing the importance of Ang II [128]. Not surprisingly, L-NAME administration results in augmentation of Ang II and its mediated effects [129]. In fact, recent reports show that plasma Ang II is elevated after just 3 weeks of treatment with L-NAME [124]. In addition to Ang II induction, the production of ROS and subsequently, oxidative stress is an important pathogenic factor in L-NAME induced hypertension [15, 74]. Vascular remodeling, fibrosis, inflammation and hypertrophy due to decreased NO, increased Ang II as well as increased ROS have all been noted in L-NAME induced hypertension [119]. Similarly to essential hypertensive states, L-NAME hypertension induces a complex, inter-connected pathophysiological response that requires an anti-hypertensive treatment capable of multiple causal and responsive factors. 18

32 Figure 1.1. N-ω-nitro-L-arginine methyl ester modulation of blood pressure. L- NAME stimulates the uncoupling of NO leading to decreased NO and consequent vasoconstriction. Activation of the RAS by L-NAME leads to increase mrna expression of renin and ACE. This results in the elevation of Ang II and it effects including increased NAD(P)H-oxidase production of super oxide anion and generation of ROS, increased aldosterone, alteration of fluid balance and activation of cytokines and chemokines. Ang II also directly stimulates vasoconstriction and increases in blood pressure. Stimulation of SNS activity results in increased heart rate, contractility and vasoconstriction. The sums of L-NAME s effects are endothelial dysfunction, remodelling and hypertrophy culminating with a hypertensive state. (Nω-nitro-L-arginine methyl ester -L-NAME; Nitric Oxide- NO; Renin andgiotensin system- RAS; ACE- angiotensin converting enzyme; Ang II- Angiotensin II; NAD(P)H- nicotinamide adenine dinucleotide phosphate; ROS- reactive oxygen species); SNS- sympathetic nervous system 19

33 1.4.2 Current therapies used to counteract L-NAME-induced hypertension Existing anti-hypertensive therapies that have been explored as potential treatments for L- NAME-induced hypertension are lacking in their ability to effectively combat all aspects of the experimental disease state, a similar predicament with anti-hypertensive therapies used to treat essential hypertension [119]. While preventing the diminished NOS activity, concurrent treatment with L-arginine, a substrate for NO synthesis, along with L-NAME did not prevent the establishment of hypertension [130]. Similarly, an anti-oxidant, N-acetylcysteine, failed to abolish hypertension, but reduced ROS production and augmented NOS activity [131]. One promising candidate for treating L-NAME-induced hypertension, hydralazine, was able to prevent the development of hypertension and restored vasodilation, but was unsuccessful in preventing the production of O 2ˉ, arterial fibrosis and inflammation [132]. Currently, chronic treatment with the ACE inhibitor, enalapril, or AT 1 - receptor blocker, losartan, showed success in combating L-NAME-induced hypertension, but both treatments still need further research to ensure abrogation of all aspects of pathophysiology involved [133]. Recently, antioxidant therapies such as curcumin and tetrahydrocurcumin significantly suppressed blood pressure elevation and oxidative stress, but did not report changes in Ang II [129]. Consequently, the lack of successful therapies available to treat hypertension, like that of L-NAME-induced hypertension, demonstrates the need for new candidates that could potentially combat all aspects of this disease. One such candidate may be the heme-l-arginate, resulting in the upregulation of the heme-oxygenase (HO) pathway. 20

34 1.5 The Heme oxygenase system Review of the Heme oxygenase system Originally discovered in 1968 by Tenhunen and Schmidt, the HO system is a powerful generator of antioxidants, anti-inflammatory molecules, and vasodilation. HO is the initial and rate-limiting, microsomal enzyme in the pathway which degrades heme [134]. There are three isoforms of HO found in the body: HO-1, HO-2 and HO-3 [135]. HO-2 and, to a lesser extent, HO-3 are constitutively expressed and regulate normal cell function [136]. Conversely, HO-1 is not normally expressed in tissues, with the exception of the spleen, bone marrow and liver (which can contain specialized reticuloendothelial cells) [137]. HO-1, a heat shock protein (HSP32), expression is induced in response to oxidative stress, ischemia-reperfusion, hypoxia, hyperthermia, tissue inflammation and by a wide array of other diverse stimuli [138]. Importantly, increases in HO-1 s enzymatic substrate (heme), promotes HO-1 protein expression and activity [136, 137]. Activation of HO-1 by its diverse array of stimulators rapidly increases widespread transcription and expression of the protein [134]. All forms of HO catalyze the oxidation of heme, a metalloporphyrin, which is a powerful oxidant and promoter of ROS generation and lipid peroxidation [139]. Free heme does not occur under normal conditions, but is deposited in tissues under pathological conditions. The catabolism of heme by HO leads to the liberation of biliverdin, carbon monoxide (CO) and free iron (in the ferrous form) through a coupled-oxidation mechanism. The coupled oxidation mechanism requires molecular oxygen and a NAD(P)H dependent reductase [134]. As such, the activity of HO not only protects cells from increased oxidation, but provides important sources of cytoprotection; CO, biliverdin, and free iron [134]. Moreover, benefits of HO protein expression are further driven by modulation of second messenger cascades such a cgmp and MAPK [140, 141]. There is a vast amount of evidence to the cytoprotective benefits of HO-1 upregulation. For instance, HO-1 regulation 21

35 protected rats from hypertensive renal damage and hypertrophy [114, 142]. While the benefits of HO-1 upregulation have been firmly established, more research must be done to clearly elucidate this pathway Carbon Monoxide First identified as a toxic air pollutant, CO is a stable, lipid soluble gas and as such cannot be held within cell membranes [143]. A bi-product from all types of incomplete combustion with carbon-containing molecules, CO has a great affinity for hemoglobin and it can reduce blood oxygenation with the potential to result in hypoxia [144]. At its normal production rate of 16.4 µmol/h with daily production totaling up to 500 µmol in humans, CO has proven to be beneficial at endogenous levels and is commonly derived as a by-product of heme oxidation by NAD(P)H [134, 144]. CO is an important vasoactive signaling gas molecule that acts similarly to NO and is involved in regulating contractility and blood pressure in vascular tissues [145]. Similar to the actions of NO, evidence shows that CO stimulates the sgc resulting in the production of cgmp [146, 147]. cgmp is involved in vasodilation as well as other vascular functions, such as inhibition of platelet aggregation and SMC proliferation [140]. Independent of cgmp, CO has been shown to activate the calcium activated potassium channel (BK Ca ) directly increasing the outward potassium (K + ) current resulting in the hyperpolarization of the SMC and vasodilation [141]. It should be noted that functional endothelium are required to carry out CO benefits [139]. While stimulating a series of vasodilatory molecules and actions, CO simultaneously inhibits endothelin, cytochrome P450 enzyme activity and 20-HETE, all of which possess inflammatory and vasoconstrictive properties [137, 148]. CO contributes to the modulation of oxidative stress by inhibiting NAD(P)H-oxidase and the subsequent production of O 2ˉ, as well as increasing glutathione levels [149, 150]. In addition to CO, biliverdin, another product from the HO 22

36 pathway which is potentially toxic and formally known merely as a waste product, has been recognized as an important cytoprotective agent Biliverdin/Bilirubin Biliverdin is a soluble greenish bile pigment which is primarily produced through the breakdown of heme by HO. It is quickly reduced to bilirubin by biliverdin reductase (BVR) [135]. Bilirubin is lipophilic, yellowish bile pigment and possesses the ability cross cell membranes [151]. Normally, the majority of bilirubin is derived from hemoglobin released from aging or damaged red blood cells [148]. This accounts for the basal expression of HO in the spleen and bone marrow. It is then conjugated and passes from the liver though bile and the feces to be excreted [134]. Interestingly, several studies have shown that elevated serum levels of bilirubin are related to a reduced risk of atherosclerosis, stroke and coronary artery disease [135, 136, 152]. Bilirubin, as a potent antioxidant and reducing agent, is able to scavenge ROS including H 2 O 2 and O 2ˉ [135, 136]. Like CO, bilirubin is able to inhibit NAD(P)H-oxidase as well as PKC activity and Ang II induced vascular damage [140, 153]. Bilirubin also decreases arterial remodeling and inflammation through the reduction of several adhesion molecules and growth factors. HO derived bilirubin exerts cytoprotective properties on the cardiovascular system, which is manifested in individuals with Gilbert syndrome whom have higher than normal serum bilirubin levels and have decreased risk for coronary artery disease [150]. Many studies have shown the benefits of physiological bilirubin levels; however, it should be noted at high concentrations bilirubin itself can generate ROS [151]. 23

37 1.5.4 Free Iron Along with CO and biliverdin, free iron is released in the catabolism of heme. The free iron released is in the ferrous form, but molecular oxygen participating in the reaction results in the conversion to the ferric form [154]. The release of free iron in the catabolism of heme could be regarded as harmful and pro-oxidant, however; this iron is not allowed to accumulate as it is rapidly sequestered by ferritin. [135]. By subsequently binding to iron regulatory protein (IRP), free iron stimulates a pathway which leads to its sequestration and exportation [134]. In particular, this exportation is due to increased ferritin synthesis, an ubiquitous intracellular protein which acts as a reservoir for excess free iron and that has been linked to the propagation of HO-1 protection [134, 155]. The increased ferritin synthesis by high free iron levels enhances iron storage capacity within the cell leading to a decrease in iron s pro-oxidant capabilities. Therefore, the expression of ferritin remains a contributing factor to the cytoprotective benefits derived from the HO system. Additionally, unique to the free form of iron, synthesis of NO occurs via induction of NFκB which promotes the induction of NOS [154]. While individually CO, biliverdin and free iron have cytoprotective properties, evidence suggests that their coordination results in maximal cellular protection through mediating vasodilation and combating inflammation and oxidative stress [156]. 1.6 Role of the heme oxygenase pathway in hypertension The potential therapeutic benefits of HO activity are of clinical interest. Over the years many studies have investigated the effects of the HO pathway on hypertension. Recently, genetic polymorphisms of HO have been implicated in human susceptibility of essential hypertension [157]. A variety of HO-1-inducers and heme substrates such as metalloporphyrins like hemin, heme-l-lysinate and heme-l-arginate have been explored as modulators of hypertension [148, 24

38 158, 159]. The induction of the HO system has proven effective in treating many forms of experimental hypertension. In fact recent evidence reveals that the HO inducer, hemin, successfully combated DOCA salt induced hypertension while increasing anti-oxidant capacity, reducing cardiac hypertrophy and a host of other related pathology associated with severe hypertension [160]. Hemin also attenuates acute phenylephrine-induced renal hypertension in stoke prone-spontaneously hypertensive rats (SP-SHR) [138]. Heme-L-arginate, in which the heme molecule is stabilized with three molecules of arginine, has been shown to be effective in lowering blood pressure in SHR [158]. In addition, research shows that HO-inducers normalize blood pressure in adult SHR with established hypertension after just three weeks of treatment [113]. Furthermore, induction of HO-1 gene expression by retroviruses result in a decrease of mean arterial pressure after introduction of Ang II [147]. Interestingly, a marked reduction of blood pressure in SHR after just four days was noted after treatment with heme-l-arginate [158]. In contrast, the absence of HO-1 expression in mice resulted in elevated blood pressure, cardiac hypertrophy and renal failure [150]. Overall, evidence suggests that HO-inducers are effective in abrogating not only hypertension, but also the pathophysiology that accompany it Implications of heme oxygenase inducers as treatment in L-NAME-induced hypertension In particular, HO-inducers have the potential to be effective in treating L-NAME-induced hypertension. While past and current proposed therapies have only partially combated the many aspects of essential hypertension, HO-inducers and their subsequent by-products may be able to treat all aspects of this complex disease. HO-inducers have the ability to cause direct vasodilation in vasculature through the release of CO which may be sufficient to negate the effect of NO inhibition and RAS caused by L-NAME [137]. Also, stimulation of cgmp leads to further 25

39 vasodilation and inhibition of NAD(P)H-oxidase and contributes to the powerful therapeutic value of HO-inducers [138, 140]. HO-inducers, including heme-l-arginate, have also successfully suppressed fibrosis and hypertrophy in animal hypertensive models [161, 162]. Importantly, heme-l-arginate reduced PLC and oxidative stress in mesenteric arteries of DOCAsalt hypertensive rats [163]. The reduced production and scavenging of ROS and free radicals, further support the hypothesis that HO-induction is a potentially viable therapy for hypertension. With the widespread distribution of HO-1, there are many benefits to be explored in the administration of heme-l-arginate including the possible reduction of a hypertensive state. 26

40 2) Rationale, Hypothesis and Objectives 2.1) Rationale Essential hypertension is a complex disease state that increases risk for morbidity and mortality. The involvement of the SNS, endothelial dysfunction and the RAS are all known to play a part in the development and maintenance of hypertension. Despite decades of research the mechanisms driving essential hypertension have yet to be fully elucidated. Studying hypertension through the use of animal models has and will continue to uncover many underlying contributors of hypertension. One such model is L-NAME induced hypertension. Similar to the pathophysiology seen in essential hypertension, L-NAME has more than just one mechanism driving its on-set of hypertension. L-NAME induces hypertension through the inhibition of NO synthesis, the stimulation of both SNS and the RAS, particularly increasing Ang II along with the formation of ROS [26, 74]. Further investigations are required to more clearly elucidate the mechanisms by which L-NAME induces hypertension. The need for an effective well rounded anti-hypertensive therapy to combat L-NAMEinduced hypertension has yet to be discovered. The role of the HO system in hypertension is continuing to be explored in many models. It has shown notable effects in SHR and DOCA animal models, not only lowering blood pressure, but ablating hypertrophy and remodeling [142, 164]. Taking these observations into account, treatment of L-NAME induced hypertension with an HO-inducer seems valid. It has been established that HO inducers promote the production of anti-inflammatory and anti-oxidative compounds through the release of CO, bilirubin and free iron from heme, effectively decreasing hypertension and its deleterious effects [148, 165]. The effects of the HO-inducer, heme-l-arginate, on Ang II levels and related oxidative stress in L- NAME-induced hypertension are unknown to date. Consequently, the mechanisms by the HO 27

41 inducer, heme-l-arginate, interacts with L-NAME-induced hypertension and its deleterious effects will be explored. 2.2) Hypothesis Administration of heme-l-arginate abrogates the development and diminishes the establishment of L-NAME-induced hypertension via upregulation of heme-oxygenase-1, the reduction of angiotensin II levels and oxidative stress. 2.3 Objectives To determine the effect of heme-l-arginate on the development of L-NAME induced hypertension To determine the effect of heme-l-arginate on the development of L-NAME induced hypertension, heme-l-arginate was administered simultaneously along with L-NAME. Vehicle groups were also included to identify any additional effects. Systolic blood pressure was measured throughout the study in conjunction with regular assessment of body weight, fluid intake and urine excretion. At 15 weeks food intake was assessed. Upon termination, heart weight was assessed. At the molecular level, the effect of heme-l-arginate on the HO system was evaluated by the determination of the protein expression of HO-1. In addition, quantification of Ang II and total antioxidant capacity was carried out to determine the effect of heme-larginate on L-NAME-induced pathology. 28

42 2.3.2 To determine the effect of heme-l-arginate on established L-NAME induced hypertension Heme-L-arginate was administered to animals with established L-NAME induced hypertension. Systolic blood pressure was measured throughout the study in conjunction with regular assessment of body weight. Upon termination, heart weight was also assessed. Similar to the first study, exploration of HO-1 protein expression, modulation of Ang II and total antioxidant capacity was done to determine the effect of heme-l-arginate on L-NAME-induced pathology. 29

43 3. Materials and methods 3.1 Animal care and handling All Animals were housed at room temperature with 12 hour light/dark cycles. Animals had ad libitum access to standard rodent chow and drinking water. This work was approved by the University of Saskatchewan Standing Committee on Animal Care and Research Ethics board and adhered to the Canadian Council on Animal Care. 3.2 Experimental Design Treatment Protocol 61 male Sprague Dawley (SD) rats, age 6 weeks, were purchased from Charles River (St. Constant, QB, Canada) and were acclimatized for 7 days in the housing facility. At 7 weeks, pretreatment systolic blood pressure was determined and body weight was recorded. Animals were divided into groups with similar mean body weight. In study one animals (L-NAME, n=10) received L-NAME, at a dose of 60 milligrams/kilogram/day (mg/kg/day) via intraperitoneal (ip) injection. In another group, (L-NAME + HA, n=13) L-NAME (60 mg/kg/day) was given simultaneously with heme-l-arginate (HA) (15 mg/kg/day) via two ip injections (Figure 3.1). Sterile deionized water, the solvent used to dissolve L-NAME, was given via ip injection, (Vehicle 1, n=6). Animals (L-NAME + Vehicle 2, n=12) received L-NAME (60 mg/kg/day) along with sterile phosphate buffered saline (PBS), the solvent used in the preparation of heme- L-arginate. All treatments above were given for 4 weeks. For study 2, the remaining 20 animals were treated with L-NAME for 4 weeks with L-NAME (60 mg/kg/day) via ip injection, after 4 weeks treatment animals were divided into two groups with similar mean body weight. Subsequently, animals continued to receive L-NAME (60 mg/kg/day) via ip injection (L-NAME, 30

44 Figure 3.1 Study timeline. Animals in study 1 were divided into 4 groups. Baseline measurements were taken at 7 weeks followed by 4 weeks of treatment for all groups. Study 1 was terminated at 11 weeks. Study was comprised of 2 groups. After baseline measuments were taken at 7 weeks, L-NAME treatment began and continued for the duration of the study. From 11 to 15 weeks, animals in the L-NAME + HA received in addition to L-NAME. N-ωnitro-L-arginine methyl ester- L-NAME; Heme-L-arginate- HA. 31